Factor VIII polymer conjugates

ABSTRACT

The invention is a proteinaceous construct comprising a Factor VIII molecule which is conjugated to a water-soluble polymer via carbohydrate moieties of Factor VIII, and methods of preparing same.

This application is a divisional application of U.S. application Ser.No. 12/184,567 filed on Aug. 1, 2008, which is a continuation-in-part ofU.S. application Ser. No. 11/729,625, filed on Mar. 29, 2007, whichclaims priority to U.S. Provisional Application Nos. 60/790,239 and60/787,968, which were filed on Jun. 6, 2006 and Mar. 31, 2006,respectively.

FIELD OF THE INVENTION

The present invention relates to a proteinaceous construct comprisingcoagulation factor VIII (FVIII) being bound to at least one watersoluble polymer, including a poly(alkylene oxide) such as polyethyleneglycol. Further the present invention relates to methods for prolongingthe in vivo-half-life of FVIII in the blood of a mammal having ableeding disorder associated with functional defects or deficiencies ofFVIII.

BACKGROUND OF THE INVENTION

Coagulation factor VIII (FVIII) circulates in plasma at a very lowconcentration and is bound non-covalently to von Willebrand factor(VWF). During hemostasis, FVIII is separated from VWF and acts as acofactor for activated factor IX (FIXa)-mediated factor X (FX)activation by enhancing the rate of activation in the presence ofcalcium and phospholipids or cellular membranes.

FVIII is synthesized as a single-chain precursor of approximately270-330 kD with the domain structure A1-A2-B-A3-C1-C2. When purifiedfrom plasma (e.g., “plasma-derived” or “plasmatic”), FVIII is composedof a heavy chain (A1-A2-B) and a light chain (A3-C1-C2). The molecularmass of the light chain is 80 kD whereas, due to proteolysis within theB domain, the heavy chain is in the range of 90-220 kD.

FVIII is also synthesized as a recombinant protein for therapeutic usein bleeding disorders. Various in vitro assays have been devised todetermine the potential efficacy of recombinant FVIII (rFVIII) as atherapeutic medicine. These assays mimic the in vivo effects ofendogenous FVIII. In vitro thrombin treatment of FVIII results in arapid increase and subsequent decrease in its procoagulant activity, asmeasured by in vitro assay. This activation and inactivation coincideswith specific limited proteolysis both in the heavy and the lightchains, which alter the availability of different binding epitopes inFVIII, e.g. allowing FVIII to dissociate from VWF and bind to aphospholipid surface or altering the binding ability to certainmonoclonal antibodies.

The lack or dysfunction of FVIII is associated with the most frequentbleeding disorder, hemophilia A. The treatment of choice for themanagement of hemophilia A is replacement therapy with plasma derived orrFVIII concentrates. Patients with severe haemophilia A with FVIIIlevels below 1%, are generally on prophylactic therapy with the aim ofkeeping FVIII above 1% between doses. Taking into account the averagehalf-lives of the various FVIII products in the circulation, this canusually be achieved by giving FVIII two to three times a week.

There are many concentrates on the market for the treatment ofhemophilia A. One of these concentrates is the recombinant productAdvate®, which is produced in CHO-cells and manufactured by BaxterHealthcare Corporation. No human or animal plasma proteins or albuminare added in the cell culture process, purification, or finalformulation of this product.

The aim of many manufacturers of FVIII concentrates and therapeuticpolypeptide drugs is to develop a next generation product with enhancedpharmacodynamic and pharmacokinetic properties, while maintaining allother product characteristics.

Therapeutic polypeptide drugs are rapidly degraded by proteolyticenzymes and neutralized by antibodies. This reduces their half-life andcirculation time, thereby limiting their therapeutic effectiveness. Theaddition of a soluble polymer or carbohydrate to a polypeptide has beenshown to prevent degradation and increase the polypeptides half-life.For instance, PEGylation of polypeptide drugs protects them and improvestheir pharmacodynamic and pharmacokinetic profiles (Harris J M et ChessR B, Nat Rev Drug Discov 2003; 2:214-21). The PEGylation processattaches repeating units of polyethylene glycol (PEG) to a polypeptidedrug. PEGylation of molecules can lead to increased resistance of drugsto enzymatic degradation, increased half-life in vivo, reduced dosingfrequency, decreased immunogenicity, increased physical and thermalstability, increased solubility, increased liquid stability, and reducedaggregation.

Thus, the addition of a soluble polymer, such as through PEGylation isone approach to improve the properties of a FVIII product. The state ofthe art is documented by different patents and patent applications:

U.S. Pat. No. 6,037,452 describes a poly(alkylene oxide)-FVIII or FIXconjugate, where the protein is covalently bound to a poly(alkyleneoxide) through carbonyl-groups of said FVIII.

EP1258497B1 describes a method to prepare conjugates of FVIII and abiocompatible polymer. This patent was supplemented by a publication ofRöstin et al. (Bioconj Chem 2000; 11:387-96). The conjugates comprise aB-domain deleted recombinant FVIII modified with monomethoxypolyethylene glycol. The conjugate had reduced FVIII function and thecoagulant activity decreased rapidly with the degree of modification.

WO04075923A3 describes polymer-FVIII molecular conjugate comprising aplurality of conjugates wherein each conjugate has one to three watersoluble polymers covalently attached to an FVIII molecule. The FVIIImolecule is B-domain-deleted.

U.S. Pat. No. 4,970,300 describes a modified FVIII, wherein an infusibleconjugate comprising a protein having FVIII activity was covalentlylinked to a nonantigenic ligand.

U.S. Pat. No. 6,048,720 describes conjugates of a polypeptide and abiocompatible polymer.

WO94/15625 describes FVIII bound to polyethylene glycol having apreferred molecular weight of no greater than 5,000 Daltons.

There remains a need for an FVIII having an attached soluble polymer toextend the half-life of the FVIII in vivo, for example, a PEGylatedFVIII, such as full-length FVIII having PEG greater than 10,000 Daltonsconjugated thereto, which retains functional activity while providing anextended half-life in vivo, as compared to non-PEGylated FVIII.

SUMMARY OF THE INVENTION

The present invention relates to a proteinaceous construct comprising aFactor VIII molecule which is conjugated to a water-soluble polymer viacarbohydrate moieties of Factor VIII, and methods of preparing same.

In one embodiment of the invention, a method of conjugating a watersoluble polymer to an oxidized carbohydrate moiety of FVIII is providedcomprising contacting the oxidized carbohydrate moiety with an activatedwater soluble polymer under conditions that allow conjugation. In arelated aspect, the water soluble polymer is selected from the groupconsisting of PEG, PSA and dextran. In still another aspect, theactivated water soluble polymer is selected from the group consisting ofPEG-hydrazide, PSA-hydrazine and aldehyde-activated dextran. In anotheraspect of the invention, the carbohydrate moiety is oxidized byincubation in a buffer comprising NaIO₄. In still another aspect of theinvention, the oxidized carbohydrate moiety of FVIII is located in the Bdomain of FVIII.

In another embodiment of the invention, a modified FVIII produced by themethod according to any of the aforementioned methods is provided. Instill another embodiment, a proteinaceous construct is providedcomprising (a) a Factor VIII molecule; and (b) at least one watersoluble polymer bound to said Factor VIII molecule, wherein the watersoluble polymer is attached to the Factor VIII via one or morecarbohydrate moieties located in the B domain Factor VIII. In a relatedaspect of the invention, the water soluble polymer is selected from thegroup consisting of PEG, PSA and dextran.

FIGURES

FIG. 1 shows the broadening and mass increase of rFVIII afterconjugation with PEG measured by SDS-PAGE with subsequentimmunoblotting.

FIG. 2 shows the pharmacokinetics of PEG-rFVIII conjugate compared tonon-conjugated FVIII in hemophilic mice. Open squares: PEGrFVIII, dose200 IU FVIII/kg. Closed diamonds: native rFVIII, dose 200 IU FVIII/kg.

FIGS. 3A-3E show the detailed analysis of PEGylation sites by SDS-PAGEusing various anti FVIII antibodies.

FIGS. 4A-4B show the thrombin-induced activation and inactivation ofnative and PEGylated rFVIII.

FIG. 5 shows the bands demonstrating the domains of native and PEGylatedrFVIII.

FIGS. 6A-6E show the extent of PEGylation of various domains of nativeand PEGylated rFVIII.

FIGS. 7A-7B show the thrombin inactivation rate of native and PEGylatedrFVIII.

DETAILED DESCRIPTION OF THE INVENTION

The invention is a proteinaceous construct comprising an FVIII moleculehaving at least a portion of the B domain intact, bound to awater-soluble polymer which include, a polyalkylene oxide, polyvinylpyrrolidone, polyvinyl alcohol, polyoxazoline, a poly acryloylmorpholineor a carbohydrate, such as polysialic acid (PSA) or dextran. In oneembodiment of the invention, the water soluble polymer is a polyethyleneglycol molecule having a molecular weight of greater than 10,000Daltons. In another embodiment, the water soluble polymer has amolecular weight of greater than 10,000 Da to about 125,000 Da, about15,000 Da to 20,000 Da, or about 18,000 Da to about 25,000 Da. In oneembodiment, the construct retains the full functional activity ofstandard therapeutic FVIII products, and provides an extended half-lifein vivo, as compared to standard therapeutic FVIII products. In anotherembodiment, the construct retains at least 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, or 150 percent (%)biological activity relative to native Factor VIII. In a related aspect,the biological activities of the construct and native Factor VIII aredetermined by the ratios of chromogenic activity to FVIII antigen value(FVIII:Chr:FVIII:Ag). In still another embodiment of the invention, thehalf-life of the construct is decreased or increased 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, or 10-foldrelative to the in vivo half-life of native Factor VIII.

The starting material of the present invention is FVIII, which can bederived from human plasma, or produced by recombinant engineeringtechniques, as described in U.S. Pat. No. 4,757,006; U.S. Pat. No.5,733,873; U.S. Pat. No. 5,198,349; U.S. Pat. No. 5,250,421; U.S. Pat.No. 5,919,766; EP 306 968.

Herein, the term “Factor VIII” or “FVIII” refers to any FVIII moleculewhich has at least a portion of the B domain intact, and which exhibitsbiological activity that is associated with native FVIII. In oneembodiment of the invention, the FVIII molecule is full-length FactorVIII. The FVIII molecule is a protein which is encoded for by DNAsequences capable of hybridizing to DNA encoding Factor VIII:C. Such aprotein may contain amino acid deletions at various sites between orwithin the domains A1-A2-B-A3-C1-C2 (U.S. Pat. No. 4,868,112). The FVIIImolecule may also be an analog of native FVIII wherein one or more aminoacid residues have been replaced by site-directed mutagenesis.

The FVIII molecules useful for the present invention include thefull-length protein, precursors of the protein, biologically active orfunctional subunits or fragments of the protein, and functionalderivatives thereof, as well as variants thereof as described hereinbelow. Reference to FVIII is meant to include all potential forms ofsuch proteins and wherein each of the forms of FVIII has at least aportion or all of the native B domain sequence intact.

According to the present invention, the term “recombinant Factor VIII”(rFVIII) may include any rFVIII, heterologous or naturally occurring,obtained via recombinant DNA technology, or a biologically activederivative thereof. In certain embodiments, the term encompassesproteins as described above and nucleic acids, encoding a rFVIII of theinvention. Such nucleic acids include, for example and withoutlimitation, genes, pre-mRNAs, mRNAs, polymorphic variants, alleles,synthetic and naturally-occurring mutants. Proteins embraced by the termrFVIII include, for example and without limitation, those proteins andpolypeptides described hereinabove, proteins encoded by a nucleic aciddescribed above, interspecies homologs and other polypeptides that: (1)have an amino acid sequence that has greater than about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% orabout 99% or greater amino acid sequence identity, over a region of atleast about 25, about 50, about 100, about 200, about 300, about 400, ormore amino acids (up to the full length sequence of 406 amino acids forthe mature native protein), to a polypeptide encoded by a referencednucleic acid or an amino acid sequence described herein; and/or (2)specifically bind to antibodies, e.g., polyclonal or monoclonalantibodies, generated against an immunogen comprising a referenced aminoacid sequence as described herein, an immunogenic fragment thereof,and/or a conservatively modified variant thereof.

Polynucleotides encoding a rFVIII of the invention include, withoutlimitation, those that (1) specifically hybridize under stringenthybridization conditions to a nucleic acid encoding a referenced aminoacid sequence as described herein, and conservatively modified variantsthereof; (2) have a nucleic acid sequence that has greater than about95%, about 96%, about 97%, about 98%, about 99%, or higher nucleotidesequence identity, over a region of at least about 25, about 50, about100, about 150, about 200, about 250, about 500, about 1000, or morenucleotides (up to the full length sequence of 1218 nucleotides of themature protein), to a reference nucleic acid sequence as describedherein.

As used herein, “endogenous FVIII” includes FVIII which originates fromthe mammal intended to receive treatment. The term also includes FVIIItranscribed from a transgene or any other foreign DNA present in saidmammal. As used herein, “exogenous FVIII” includes FVIII which does notoriginate from said mammal.

Variant (or analog) polypeptides include insertion variants, wherein oneor more amino acid residues are added to an FVIII amino acid sequence ofthe invention. Insertions may be located at either or both termini ofthe protein, and/or may be positioned within internal regions of theFVIII amino acid sequence. Insertion variants, with additional residuesat either or both termini, include for example, fusion proteins andproteins including amino acid tags or other amino acid labels. In oneaspect, the FVIII molecule may optionally contain an N-terminal Met,especially when the molecule is expressed recombinantly in a bacterialcell such as E. coli.

In deletion variants, one or more amino acid residues in a FVIIIpolypeptide as described herein are removed. Deletions can be effectedat one or both termini of the FVIII polypeptide, and/or with removal ofone or more residues within the FVIII amino acid sequence. Deletionvariants, therefore, include all fragments of a FVIII polypeptidesequence.

In substitution variants, one or more amino acid residues of a FVIIIpolypeptide are removed and replaced with alternative residues. In oneaspect, the substitutions are conservative in nature and conservativesubstitutions of this type are well known in the art. Alternatively, theinvention embraces substitutions that are also non-conservative.Exemplary conservative substitutions are described in Lehninger,[Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp.71-77] and set out immediately below.

CONSERVATIVE SUBSTITUTIONS SIDE CHAIN CHARACTERISTIC AMINO ACIDNon-polar (hydrophobic): A. Aliphatic A L I V P B. Aromatic F W C.Sulfur-containing M D. Borderline G Uncharged-polar: A. Hydroxyl S T YB. Amides N Q C. Sulfhydryl C D. Borderline G Positively charged (basic)K R H Negatively charged (acidic) D EAlternatively, exemplary conservative substitutions are set outimmediately below.

CONSERVATIVE SUBSTITUTIONS II EXEMPLARY ORIGINAL RESIDUE SUBSTITUTIONAla (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, ArgAsp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys,Arg Ile (I) Leu, Val, Met, Ala, Phe, Leu (L) Ile, Val, Met, Ala, Phe Lys(K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro(P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, SerVal (V) Ile, Leu, Met, Phe, Ala

A “naturally-occurring” polynucleotide or polypeptide sequence istypically from a mammal including, but not limited to, primate, e.g.,human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or anymammal. The nucleic acids and proteins of the invention can berecombinant molecules (e.g., heterologous and encoding the wild typesequence or a variant thereof, or non-naturally occurring). Referencepolynucleotide and polypeptide sequences include, e.g.,UniProtKB/Swiss-Prot P00451 (FA8_HUMAN); Gitschier J et al.,Characterization of the human Factor VIII gene, Nature, 312(5992):326-30 (1984); Vehar G H et al., Structure of human Factor VIII, Nature,312(5992):337-42 (1984); and Thompson A R. Structure and Function of theFactor VIII gene and protein, Semin Thromb Hemost, 2003:29; 11-29(2002), (references incorporated herein in their entireties).

As used herein “biologically active derivative” or “biologically activevariant” includes any derivative or variant of a molecule havingsubstantially the same functional and/or biological properties of saidmolecule, such as binding properties, and/or the same structural basis,such as a peptidic backbone or a basic polymeric unit.

As used herein, “plasma-derived FVIII” or “plasmatic” includes all formsof the protein found in blood obtained from a mammal having the propertyof activating the coagulation pathway.

In various aspects, production of rFVIII includes any method known inthe art for (i) the production of recombinant DNA by geneticengineering, (ii) introducing recombinant DNA into prokaryotic oreukaryotic cells by, for example and without limitation, transfection,electroporation or microinjection, (iii) cultivating said transformedcells, (iv) expressing rFVIII, e.g. constitutively or upon induction,and (v) isolating said rFVIII, e.g. from the culture medium or byharvesting the transformed cells, in order to (vi) obtain purifiedrFVIII.

In other aspects, the rFVIII is produced by expression in a suitableprokaryotic or eukaryotic host system characterized by producing apharmacologically acceptable rFVIII molecule. Examples of eukaryoticcells are mammalian cells, such as CHO, COS, HEK 293, BHK, SK-Hep, andHepG2.

In still other aspects, a wide variety of vectors are used for thepreparation of the rFVIII and are selected from eukaryotic andprokaryotic expression vectors. Examples of vectors for prokaryoticexpression include plasmids such as, and without limitation, pRSET, pET,and pBAD, wherein the promoters used in prokaryotic expression vectorsinclude one or more of, and without limitation, lac, trc, trp, recA, oraraBAD. Examples of vectors for eukaryotic expression include: (i) forexpression in yeast, vectors such as, and without limitation, pAO, pPIC,pYES, or pMET, using promoters such as, and without limitation, AOX1,GAP, GAL1, or AUG1; (ii) for expression in insect cells, vectors such asand without limitation, pMT, pAc5, pIB, pMIB, or pBAC, using promoterssuch as and without limitation PH, p10, MT, Ac5, OpIE2, gp64, or polh,and (iii) for expression in mammalian cells, vectors such as and withoutlimitation pSVL, pCMV, pRc/RSV, pcDNA3, or pBPV, and vectors derivedfrom, in one aspect, viral systems such as and without limitationvaccinia virus, adeno-associated viruses, herpes viruses, orretroviruses, using promoters such as and without limitation CMV, SV40,EF-1, UbC, RSV, ADV, BPV, and β-actin.

In certain aspects, FVIII molecules are conjugated to a water solublepolymer by any of a variety of chemical methods (Roberts J M et al.,Advan Drug Delivery Rev 2002; 54:459-76). For example, in one embodimentFVIII is PEGylated by the conjugation of PEG to free amino groups of theprotein using N-hydroxysuccinimide (NHS) esters. In another embodimentthe water soluble polymer, for example PEG, is coupled to free SH groupsusing maleimide chemistry or the coupling of PEG hydrazides or PEGamines to carbohydrate moieties of the FVIII after prior oxidation.

In other embodiments, FVIII is conjugated to other water solublepolymers, where the water soluble polymers are, for example,polyalkylene oxide, polyvinyl pyrrolidone, polyvinyl alcohol,polyoxazoline, a poly acryloylmorpholine, carbohydrate or apolysaccharide such as polysialic acid (PSA) or dextran. The coupling ofthe water soluble polymer can be carried out by direct coupling to theprotein or via linker molecules. One example of a chemical linker isMBPH (4-[4-N-Maleimidophenyl]butyric acid hydrazide) containing acarbohydrate-selective hydrazide and a sulfhydryl-reactive maleimidegroup (Chamow et al., J Biol Chem 1992; 267:15916-22).

The conjugation can be performed by direct coupling (or coupling vialinker systems) of the water soluble polymer to Factor VIII underformation of stable bonds. In addition degradable, releasable orhydrolysable linker systems can be used in the present invention(Tsubery et al. J Biol Chem 2004; 279:38118-24/Greenwald et al., J MedChem 1999; 42:3657-67/Zhao et al., Bioconj Chem 2006;17:341-51/WO2006/138572A2/U.S. Pat. No. 7,259,224B2/U.S. Pat. No.7,060,259B2).

As discussed herein, an embodiment of the invention is the coupling ofthe activated soluble polymer to the oxidized carbohydrate moiety ofFVIII. The term “activated water soluble polymer” is used herein torefer to water soluble polymers used for coupling to FVIII having anactive functional group, which allows chemical conjugation of the watersoluble polymer to a linker or directly to FVIII (which contains anactive aldehyde group). The term “oxidized carbohydrate moiety” as usedherein refers to FVIII containing free aldehyde groups, which aregenerated by an oxidative agent such as NaIO₄. In one aspect of theinvention, aldehyde-activated dextran (containing an active aldehydegroups) is coupled to the aldehyde groups of FVIII via a dihydrazidelinker.

According to the glycosylation pattern of FVIII (Lenting et al; Blood,92:3983-96 (1998)), conjugation of FVIII via carbohydrate moietiesshould likely take place in the B domain of FVIII. Targeting the Bdomain for such conjugation reactions is desired since the B domain doesnot play a role in the activity of FVIII. Enzymatic glycoconjugation isdescribed in US 2008/00700275.

In one embodiment of the invention, FVIII was modified via lysineresidues by use of polyethylene glycol derivatives containing an activeN-hydroxysuccinimide ester (NHS) such as succinimidyl succinate,succinimidyl glutarate or succinimidyl propionate. These derivativesreact with the lysine residues of FVIII under mild conditions by forminga stable amide bond. In one embodiment of the invention, the chainlength of the PEG derivative is 5,000 Da. Other PEG derivatives withchain lengths of 500 to 2,000 Da, 2,000 to 5,000 Da, greater than 5,000up to 10,000 Da or greater than 10,000 up to 20,000 Da, or greater than20,000 up to 150,000 Da are used in various embodiments, includinglinear and branched structures.

Alternative methods for the PEGylation of amino groups are the chemicalconjugation with PEG carbonates by forming urethane bonds, or thereaction with aldehydes or ketones by reductive amination formingsecondary amide bonds.

In the present invention an FVIII molecule is chemically modified usingPEG derivatives that are commercially available. These PEG derivativescan have a linear or branched structures. Examples of PEG-derivativescontaining NHS groups are listed below.

The following PEG derivatives are examples of those commerciallyavailable from Nektar Therapeutics (Huntsville, Ala.; seewww.nektar.com/PEG reagent catalog; Nektar Advanced PEGylation, pricelist 2005-2006):

mPEG-Succinimidyl propionate (mPEG-SPA)

mPEG-Succinimidyl α-methylbutanoate (mPEG-SMB)

mPEG-CM-HBA-NHS (CM=carboxymethyl; HBA=Hydroxy butyric acid)

Structure of a Branched PEG-Derivative (Nektar Therapeutics):

Branched PEG N-Hydroxysuccinimide (mPEG2-NHS)

This reagent with branched structure is described in more detail byKozlowski et al. (BioDrugs 2001; 5:419-29).

Other examples of PEG derivatives are commercially available from NOFCorporation (Tokyo, Japan; see www.nof.co.jp/english: Catalogue 2005)

General Structure of Linear PEG-Derivatives (NOF Corp.):

X=carboxymethyl

X=carboxypentyl

x=succinate

x=glutarate

Structures of Branched PEG-Derivatives (NOF Corp.):2,3-Bis(methylpolyoxyethylene-oxy)-1-(1,5-dioxo-5-succinimidyloxy,pentyloxy)propane

2,3-Bis(methylpolyoxyethylene-oxy)-1-(succinimidylcarboxypentyloxy)propane

These propane derivatives show a glycerol backbone with a 1,2substitution pattern. In the present invention branched PEG derivativesbased on glycerol structures with 1,3 substitution or other branchedstructures described in US2003/0143596A1 can also be used.

PEG derivatives with degradable (for example, hydrolysable linkers) asdescribed by Tsubery et al. (J Biol Chem 2004; 279:38118-24) andShechter et al. (WO04089280A3) can also be used in the presentinvention.

Surprisingly, the PEGylated FVIII of this invention exhibits fullfunctional activity, combined with an extended FVIII half-life in vivo.In addition the PEGylated rFVIII seems to be more resistant againstthrombin inactivation. This was shown by a variety of in vitro and invivo methods, and is illustrated by the following examples.

As used herein, “sialic acid moieties” includes sialic acid monomers orpolymers (“polysaccharides”) which are soluble in an aqueous solution orsuspension and have little or no negative impact, such as side effects,to mammals upon administration of the PSA-FVIII-conjugate in apharmaceutically effective amount. There is no particular limitation tothe sialic acid unit used according to the present invention. Thepolymers are characterized, in one aspect, as having from 1 to 4 units.In certain aspects, different sialic acid units are combined in a chain.

In various aspects of the invention, sialic acid moieties are bound toFVIII for example by the method described in U.S. Pat. No. 4,356,170,which is herein incorporated by reference. In various embodiments of theinvention, the polysaccharide compound is a naturally occurringpolysaccharide, a derivative of a naturally occurring polysaccharide, ora naturally occurring polysaccharide derivative. Generally, all of thesaccharide residues in the compound are sialic acid residues.

Other techniques for coupling PSA to polypeptides are also known. Forexample, US Publication No. 2007/0282096 describes conjugating an amineor hydrazide derivative of, e.g., PSA, to proteins. In addition, USPublication No. 2007/0191597 describes PSA derivatives containing analdehyde group for reaction with substrates (e.g., proteins) at thereducing terminal end.

In one embodiment of the invention, the polysialic acid portion of thepolysaccharide compound is highly hydrophilic, and in another embodimentthe entire compound is highly hydrophilic. Hydrophilicity is conferredprimarily by the pendant carboxyl groups of the sialic acid units, aswell as the hydroxyl groups. The saccharide unit may contain otherfunctional groups, such as, amine, hydroxyl or sulphate groups, orcombinations thereof. These groups may be present on naturally occurringsaccharide compounds, or introduced into derivative polysaccharidecompounds.

Polysaccharide compounds of particular use for the invention are, in oneaspect. those produced by bacteria. Some of these naturally occurringpolysaccharides are known as glycolipids. In one embodiment, thepolysaccharide compounds are substantially free of terminal galactoseunits.

In one embodiment of the present invention, the in vivo half-life of theproteinaceous construct is prolonged. In a related embodiment, the invivo half-life of the proteinaceous construct is prolonged by at least afactor of two, while in another embodiment the in vivo half-life isprolonged by at least a factor of three, as compared to FVIII which isnot bound to a water soluble polymer.

In one embodiment the proteinaceous construct of the present inventionmay be administered by injection, such as intravenous, intramuscular, orintraperitoneal injection.

To administer compositions comprising a proteinaceous construct of thepresent invention to human or test animals, in one aspect, thecompositions comprise one or more pharmaceutically acceptable carriers.The terms “pharmaceutically” or “pharmacologically acceptable” refer tomolecular entities and compositions that are stable, inhibit proteindegradation such as aggregation and cleavage products, and in additiondo not produce allergic, or other adverse reactions when administeredusing routes well-known in the art, as described below.“Pharmaceutically acceptable carriers” include any and all clinicallyuseful solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents and the like,including those agents disclosed above.

As used herein, “effective amount” includes a dose suitable for treatinga mammal having a bleeding disorder as outlined above.

The compositions may be administered orally, topically, transdermally,parenterally, by inhalation spray, vaginally, rectally, or byintracranial injection. The term parenteral as used herein includessubcutaneous injections, intravenous, intramuscular, intracisternalinjection, or infusion techniques. Administration by intravenous,intradermal, intramuscular, intramammary, intraperitoneal, intrathecal,retrobulbar, intrapulmonary injection and or surgical implantation at aparticular site is contemplated as well. Generally, compositions areessentially free of pyrogens, as well as other impurities that could beharmful to the recipient.

Single or multiple administrations of the compositions can be carriedout with the dose levels and pattern being selected by the treatingphysician. For the prevention or treatment of disease, the appropriatedosage will depend on the type of disease to be treated, as describedabove, the severity and course of the disease, whether drug isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the drug, and thediscretion of the attending physician.

The present invention also relates to a pharmaceutical compositioncomprising an effective amount of a proteinaceous construct as definedabove. The pharmaceutical composition may further comprise apharmaceutically acceptable carrier, diluent, salt, buffer, orexcipient. The pharmaceutical composition can be used for treating theabove-defined bleeding disorders. The pharmaceutical composition of theinvention may be a solution or a lyophilized product. Solutions of thepharmaceutical composition may be subjected to any suitablelyophilization process.

As an additional aspect, the invention includes kits which comprise acomposition of the invention packaged in a manner which facilitates itsuse for administration to subjects. In one embodiment, such a kitincludes a compound or composition described herein (e.g., a compositioncomprising a proteinaceous construct), packaged in a container such as asealed bottle or vessel, with a label affixed to the container orincluded in the package that describes use of the compound orcomposition in practicing the method. In one embodiment, the kitcontains a first container having a composition comprising aproteinaceous construct and a second container having a physiologicallyacceptable reconstitution solution for the composition in the firstcontainer. In one aspect, the compound or composition is packaged in aunit dosage form. The kit may further include a device suitable foradministering the composition according to a specific route ofadministration. Preferably, the kit contains a label that describes useof the therapeutic protein or peptide composition.

EXAMPLES Example 1 PEGylation of Lysine Residues in rFVIII with mPEGSuccinimidyl Succinate

A solution of a rFVIII bulk derived from the Advate manufacturingprocess (3,400 U/ml) was gel filtrated by use of Econo-Pac 10DG columns(Bio-Rad) using 20 mM Hepes buffer, 150 mM NaCl, pH 7.4, containing 0.5%sucrose and 0.1% Polysorbate 80. Then mPEG Succinimidyl succinate(Abuchowski et al. Cancer Biochim Biophys 1984; 7:175-86) with a chainlength of 5,000 Da (PEG-SS 5000) was added to this solution under gentlestirring (5 mg PEG-SS/mg protein) and the pH value was adjusted to 7.4by drop wise addition of 0.5 M NaOH. Then the PEGylation was carried outunder gentle stirring for 1 hour at room temperature.

Subsequently the reaction mixture was applied onto an equilibratedion-exchange chromatography resin (Fractogel EMD TMAE 650M/PharmaciaXK-10 column, bed height: 15.0 cm) in 20 mM Hepes buffer, 150 mM NaCl,pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80. Then the columnwas washed with 20 CV equilibration buffer to remove excess reagent andthe PEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 MNaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4). The eluate wasconcentrated by ultrafiltration/diafiltration with a membrane consistingof regenerated cellulose and with a molecular weight cut-off of 30 kDusing a buffer system consisting of 20 mM Hepes, 150 mM NaCl, 0.5%sucrose, pH 7.4.

Example 2 Biochemical Characterization of PEGylated rFVIII in vitro

RFVIII derived from the Advate manufacturing process was PEGylatedaccording to Example 1 and the PEGylated FVIII product was biochemicallycharacterized. The functional activity of the PEG-rFVIII was determinedby use of the FVIII chromogenic assay (Rosen S, Scand J Haematol 1984;33 (Suppl 40):139-45). The method is based on Ph. Eur. 5th edition(5.05) 2.7.4 Assay of Blood Coagulation Factor VIII.

A sample, containing factor VIII (FVIII:C) is mixed with thrombin,activated factor IX (FIXa), phospholipids and factor X (FX) in a buffercontaining calcium. FVIII is activated by thrombin and subsequentlyforms a complex with phospholipids, FIXa and calcium ions. This complexactivates factor X to factor Xa, which in turn cleaves the chromogenicsubstrate FXa-1 (AcOH*CH3OCO-D-CHA-Gly-Arg-pNA). The time course ofpara-nitroaniline (pNA) released is measured with a micro plate readerat 405 nm. The slope of the reaction is proportional to the factor VIIIconcentration in the sample. The FVIII antigen value was measured by useof an ELISA system commercially available (Cedarlane, Hornby, Ontario,Canada) with minor modifications. From these values the ratios FVIIIchromogen/FVIII antigen were calculated. The protein content in thepreparations was determined by measuring the optical density at 280 nm.From these data the protein content was calculated (Hoyer L W in: HumanProtein Data. Installments 1-6; Heberli Ed.; Wiley V C H, Weinheim,Germany, 1998) and expressed in mg/ml.

TABLE 1 PEG-rFVIII Native PEG-SS 5K rFVIII (5 mg per mg protein)FVIII:Chr activity 3,430 64 [U/ml] FVIII:Ag 4,067 81 [U/ml] Ratio 0.840.79 FVIII:Chr/FVIII:Ag Recovery of biological 100 94 activity (%)

The data in Table 1 shows that in the PEGylated rFVIII preparation, thebiological activity (expressed by the ratio FVIII chromogenic activityto FVIII antigen) is recovered to more than 90% in comparison to thebiological activity of the native rFVIII (100%).

Example 3 Characterization of PEGylated rFVIII by SDS-PAGE andImmunoblotting Techniques

Native rFVIII was characterized by SDS PAGE under reducing conditions byusing a 4-12% polyacrylamide gradient gel obtained from Invitrogen(Carlsbad, Calif., USA) according to the instructions of themanufacturer. As molecular weight markers (MW) Precision Plus markers(10 kD-250 kD) obtained from Bio-Rad (Hercules, Calif., USA) were used.Then the proteins were transferred on a PVDF membrane obtained fromBio-Rad (Hercules, Calif., USA) by electroblotting and subsequentlyincubated with a polyclonal sheep anti human FVIII:C antibody obtainedfrom Cedarlane (Hornby, Ontario, Canada). The last steps of theimmunostaining procedure were the incubation with an alkalinephosphatase (ALP) conjugated anti-sheep antibody obtained from Accurate(Westbury, N.Y., USA) followed by the final visualization by use of anALP substrate kit (Bio-Rad, Hercules, Calif., USA). The results aresummarized in FIG. 1. The blot demonstrates the domain structure ofnative and PEGylated rFVIII. It is shown that the PEGylated rFVIII hasbroader bands and high molecular masses than the native recombinantprotein.

Example 4 Pharmacokinetics of PEGylated rFVIII in a FVIII DeficientKnock Out Mouse Model

FVIII deficient mice described in detail by Bi et al. (Nat Genet 1995;10:119-21) were used as a model of severe human hemophilia A. Groups of5 mice received a bolus injection (10 ml/kg) via the tail vein witheither PEG-rFVIII (PEG-SS, 5K) prepared according to Example 1 or nativerFVIII in a dose of 200 IU FVIII/kg bodyweight. Citrate plasma by heartpuncture after anesthesia was prepared from the respective groups, 5minutes, 3, 6, 9 and 24 hours after injection. FVIII activity levelswere measured in plasma samples. The results of this experiment aresummarized in FIG. 2. Mean half life increased from 1.9 hours (fornative rFVIII) to 4.9 hours (for PEGylated rFVIII), area under curve(AUC) increased from 13.0 to 25.2 hours*IU/ml. Half-life calculation wasperformed with MicroMath Scientist, model 1 from pharmacokinetic library(MicroMath, Saint Louis, Mo., USA).

Example 5 Detailed Analysis of PEGylation of rFVIII by SDS-PAGE andImmunoblotting Techniques

Native and PEGylated rFVIII was digested with 1 nM thrombin for 60minutes at 60° C., which resulted in specific cleavage of the FVIIImolecule with well defined degradation products. These heavy- and lightchain fragments were separated by SDS-PAGE followed by electroblotting,as described in Example 3. To visualize the cleaved fragments, apolyclonal antibody and monoclonal antibodies against the heavy chain A1and A2 domains, the B domain and the light chain N-terminal A3 domainwere applied.

As seen in FIG. 3 all domains were PEGylated, albeit to a differentextent. The B domain was strongly PEGylated. Both the A1 and A2 domainsof the heavy chain were partially PEGylated. Various PEGylation-degrees(mono-, di-, tri- . . . ) could be observed in the light chainA3-domain. In agreement with Example 6, the PEGylated FVIII seemed to bemore resistant to thrombin.

Example 6 Thrombin-Resistancy of PEGylated rFVIII

In vitro thrombin treatment of FVIII results in a rapid increase andsubsequent decrease in its procoagulant activity. The rate of activationand inactivation, which depends on the thrombin concentration and on theintegrity of FVIII, was monitored by a FIXa cofactor assay, as follows:

FVIII was incubated at 37° C. with 0.5 or 1 nM thrombin. Subsamples werewithdrawn at time intervals between 0.5 to 40 minutes and added to amixture of FIXa, FX, PL-vesicles and CaCl₂ also containing a specificthrombin inhibitor to stop the further thrombin-mediated reactions andincubated for 3 minutes. A subsample was added to a chromogenicsubstrate, which is selectively cleaved by FXa and contained EDTA tostop further Xa activation. After a 15 min incubation, the reaction wasterminated by acetic acid. The absorbance (A405) values, which areproportional to the FXa concentrations, were measured in an ELISA readerand converted to FXa concentrations using a purified FXa referencecurve. The generated FXa concentrations were plotted against theincubation time with thrombin.

Pseudo-first order inactivation rate of FVIII was determined by fittingthe declining part of the curves with a single exponential fit.

TABLE 2 First order inactivation Rate k′ (1/min) Native Relative k′Thrombin FVIII PEG-FVIII PEG/native 0.5 nM 0.14 0.08 0.57   1 nM 0.240.14 0.58

As shown in FIG. 4 and Table 2, PEGylated rFVIII showed a slowerinactivation rate at both applied thrombin concentrations.

Example 7 PEGylation of Lysine Residues in rFVIII with Branched2,3-Bis(methylpolyoxyethylene-oxy)-1-(1,5-dioxo-5-succinimidyloxy,pentyloxy)propane

A solution of rFVIII in 20 mM Hepes buffer pH 7.4 containing 150 mMNaCl, 0.5% sucrose and 0.1% Polysorbate 80 was prepared from bulkmaterial derived from the Advate manufacturing process containing 489 IUFVIII/ml. A branched PEG succinimidyl glutarate (PEG-SG) reagent(2,3-Bis(methylpolyoxyethylene-oxy)-1-(1,5-dioxo-5-succinimidyloxy,pentyloxy)propane) obtained from NOF Corporation (Tokyo, Japan) with amolecular weight of 20 kD was added to 153 ml of this solution undergentle stirring (5 mg reagent/mg protein) and the pH value was adjustedto 7.4 by drop wise addition of 0.5 M NaOH after 10 minutes. Then thePEGylation of rFVIII was performed under gentle stirring for 1 hour atroom temperature.

Subsequently the reaction mixture was applied onto an equilibratedion-exchange chromatography resin (Fractogel EMD TMAE 650M/PharmaciaXK-50 column, bed height: 14.5 cm) in 20 mM Hepes buffer, 150 mM NaCl,pH 7.4, containing 0.5% sucrose and 0.1% Polysorbate 80 using a linearflow rate of 1 cm/min. The column was washed with 25 CV equilibrationbuffer to remove excess reagent (linear flow rate: 2 cm/min) and thePEGylated rFVIII was eluted with elution buffer (20 mM Hepes, 1.0 MNaCl, 0.5% sucrose, 0.1% Polysorbate 80, pH 7.4) at a linear flow rateof 0.5 cm/min. Then the eluate was concentrated byultrafiltration/diafiltration with a membrane consisting of regeneratedcellulose and with a molecular weight cut-off of 30 kD using a buffersystem consisting of 20 mM Hepes, 150 mM NaCl, 0.5% sucrose, pH 7.4.

Example 8 In-Vitro Characterization of rFVIII PEGylated with BranchedPEG-SG 20 kD

RFVIII derived from the Advate manufacturing process was PEGylated vialysine residues using a branched PEG-SG reagent according to Example 7and the PEGylated rFVIII product was biochemically characterized asdescribed in Example 2.

TABLE 3 PEG-rFVIII Native PEG-SG 20K rFVIII (5 mg per mg protein)FVIII:Chr activity 9,950 1,040 [U/ml] FVIII:Ag 20,807 1,763 [U/ml] Ratio0.48 0.59 FVIII:Chr/FVIII:Ag Recovery of biological 100 120 activity (%)

The data in Table 3 show that in the PEGylated rFVIII preparation thebiological activity (expressed by the ratio FVIII chromogenic activityto FVIII antigen) completely recovered in comparison to the biologicalactivity of the native rFVIII (100%).

The PEGylated rFVIII was characterized by SDS-PAGE and immunoblottingtechniques under reducing conditions using a 4-12% polyacrylamidegradient gel as described in Example 3. The results are summarized inFIG. 5. The blot demonstrates the domain structure of native andPEGylated rFVIII. It is shown that the PEGylated rFVIII has broaderbands and high molecular masses than the native recombinant protein.

For more detailed analysis of PEGylation of the rFVIII preparation bySDS-PAGE and immunoblotting techniques, the native and PEGylated rFVIIIwas digested with 1 nM thrombin for 60 minutes at 60°, which resulted inspecific cleavage of the FVIII molecule with well defined degradationproducts, as described in Example 5. The fragments were separated bySDS-PAGE followed by electroblotting and visualized by differentanti-FVIII antibodies. As seen in FIG. 6, all domains were PEGylated,albeit to a different extent. The B domain was strongly PEGylated.Various PEGylation-degrees (mono-, di-, tri-PEGylation) could beobserved in the light chain A3-domain. The results indicate that thePEGylated rFVIII seemed to be more resistant to thrombin.

The rate of activation and inactivation by thrombin was monitored by aFIXa cofactor assay as described in Example 6. Pseudo-first orderinactivation rate of FVIII was determined by fitting the declining partof the curves with a single exponential fit.

TABLE 4 First order inactivation Rate k′ (1/min) Relative k′ ThrombinNative FVIII PEG-FVIII PEG/native 0.5 nM 0.13 0.09 0.67   1 nM 0.21 0.150.71

As shown in FIG. 7 and Table 4, the PEGylated rFVIII showed a slowerinactivation rate at both applied thrombin concentrations.

Example 9 PEGylation of rFVIII Via Carbohydrate Moiety

For preparation of a PEG-rFVIII conjugate via carbohydrate residues, asolution of rFVIII (final concentration: 1.2 mg/ml) is prepared in 25 mMphosphate buffer, pH 6.7. NaIO₄ is added (final concentration 0.3 mM)for the oxidation of carbohydrate residues (Roberts et al.; AdvancedDrug Del Rev.; 54:459-76 (2002); Meir and Wilchek; Meth Enzymol; 138:429-42 (1987)). The reaction was quenched by addition of glycerol in afinal concentration of 10%, and the excess reagents were separated byrepeated centrifugation using Amicon Micron-10 devices (Amicon,Billerica, Mass.). PEG-hydrazide (MW 3300 Da/Nektar, Huntsville, Ala.)was added to give a final concentration of 1.5 mM reagent. ThePEGylation was then performed for 2 h at room temperature. Subsequently,the conjugate obtained and the excess reagent was separated by repeatedcentrifugation on Amicon Micron-10 devices using 25 mM phosphate buffer,pH 6.7.

Example 10 Polysialylation of rFVIII with PSA-Hydrazine

For preparation of a PSA-rFVIII conjugate via carbohydrate residues, asolution of rFVIII (final concentration: 1 mg/ml) is prepared in 20 mMsodium acetate buffer, pH 6.0. NaIO₄ is added (final concentration 0.25mM) for the oxidation of carbohydrate residues. The oxidation is carriedout for 60 min at 4° C. in the dark. Sodium bisulfite (finalconcentration 25 mM) is added to stop the reaction. The excess sodiumperiodate is separated by gelfiltration on DG-10 columns (Bio-Rad).Subsequently, PSA-hydrazine with a chain length of 20 kD (preparedaccording to WO2006/016168) is added (final concentration 10 mM). Thepolysialylation procedure is carried out for 2 h at room temperature.The polysialylated rFVIII is purified by HIC on Butyl-Sepharose(GE-Healthcare). A 5 M NaCl solution is added to the mixture to give afinal concentration of 3M NaCl. This mixture is applied to the columnfilled with Butyl-Sepharose (GE-Healthcare) and the elution of therFVIII-PSA conjugate is carried out with 50 mM Hepes-buffer, pH 7.4,containing 6.7 mM CaCl₂. After elution of the conjugate, the pH isadjusted to pH 6.9.

Example 11 Purification and Derivatization of Polysialic Acid

Polysialic Acid was purified by anion-exchange chromatography onQ-Sepharose FF as described in WO06016161A1. Five grams of PSA weredissolved in 50 mL 10 mM Triethanolamine buffer, pH 7.4 containing 25 mMNaCl (=starting buffer). This solution was applied onto a Pharmacia XK50column filled with Q-Sepharose FF (GE Healthcare, Munich, Germany),which was equilibrated with starting buffer. The column was next washedwith 8 column volumes (CV) starting buffer and the bound PSA was elutedstepwise with 3CV 200 mM NaCl, 350 mM NaCl and 500 mM NaCl in startingbuffer. The fraction eluted with 350 mM NaCl showed a molecular weightof 20 kDa as indicated by SDS gel electrophoresis. This fraction wasconcentrated by ultrafiltration using a 5 kD membrane made ofregenerated cellulose (Millipore, Billerica, Mass.) and subsequentlydiafiltrated against 50 mM phosphate buffer, pH 7.2. The PSA wasoxidized with NaIO₄ and a terminal primary amino group was introduced byreductive amination as described in WO05016973A1. For reductiveamination, 11 mL of a 2 M NH₄Cl solution were added to 20 mL of asolution containing 58 mg oxidized PSA/ml in 50 mM phosphate buffer, pH7.2. A solution of 5M NaCNBH₃ in 1M NaOH was then added to give a finalconcentration of 75 mM. The reaction was performed for 5 d at roomtemperature at pH 8.0.

The mixture was then dialyzed against a (NH₄)₂CO₃ solution (50 mg/L)containing 10 mM NaCl and subsequently against 50 mM phosphate buffer,pH 8.0, containing 5 mM EDTA. A sulfhydryl group was next introduced byreaction of the terminal primary amino group with 2-iminothiolane(Traut's reagent/Pierce, Rockford, Ill.). The reaction was carried outin 50 mM phosphate buffer, pH 8.0, containing 5 mM EDTA with 20 foldmolar excess of reagent for 1 h at room temperature. Finally the PSAsolution containing a terminal free SH-group was subjected toultrafiltration/diafiltration using a membrane with a cut-off of 5 kDand made of regenerated cellulose (Millipore, Billerica, Mass.).

Example 12 Polysialylation of rFVIII by use of a HeterobifunctionalCross-Linker

For coupling of PSA-SH to rFVIII, the heterobifunctional cross-linkerMBPH (4-[4-N-Maleimidophenyl]butyric acid hydrazide.HCl/Pierce,Rockford, Ill.) containing a carbohydrate-selective hydrazide and asulfhydryl-reactive maleimide group was used (Chamow et al., J BiolChem; 267:15916-22 (1992)). PSA-SH containing an active sulfhydryl groupwas prepared according to Example 11.

Two ml rFVIII (638 mg, 3.856 mg/ml protein concentration) weretransferred to oxidation buffer (50 mM sodium acetate, pH 6) usingdesalting columns (Bio-Rad Econopac 10 DG) according to the instructionsof the manufacturer. The protein was then oxidized with 0.25 mM NaIO₄(Merck) (1 h at 4° C. in the dark). The oxidation reaction was quenchedwith glycerol in a final concentration of 10%. Glycerol and NaIO₄ wereremoved and the protein was transferred into reaction buffer (50 mMsodium phosphate pH 6.5) using desalting columns (Bio-Rad Econopac 10DG) according to the manufacturer's instructions. A mixture containing 1mg MBPH/mg protein and PSA-SH (200 fold molar excess to protein) werenext incubated for 2 h at RT at pH 6.5. The excess of linker was removedusing desalting columns (Bio-Rad Econopac 10 DG) according to theinstructions of the manufacturer and the linker-PSA conjugate wastransferred into reaction buffer.

The MPBH-PSA conjugate was added to the oxidized rFVIII (0.105 mg/mlprotein) and the reaction mixture was incubated for 2 h at RT undergentle shaking. The rFVIII-PSA conjugate was purified by HIC using aprepacked Butyl Sepharose column (GE Healthcare, Butyl HiTrap FF 5 ml).To allow hydrophobic interactions of the conjugate with Butyl Sepharosethe sample was cooled to 2-8° C. and the ionic strength of the reactionmixture was increased to a conductivity of approx. 185 mS/cm by adding abuffer solution containing 5 M NaCl (50 mM Hepes, 5 M NaCl, 6.7 mMCaCl₂, 0.01% Tween, pH 6.9). The reaction mixture was loaded onto thecolumn that was equilibrated with equilibration buffer pH 6.9(containing 50 mM Hepes, 3 M NaCl, 6.7 mM CaCl₂, 0.01% Tween 80) with aflow rate of 1.2 cm/min. Unbound sample was washed out with 10 columnvolumes (CV) of equilibration buffer. The conjugate was eluted with abuffer of low ionic strength, pH 7.4 (50 mM Hepes, 6.7 mM CaCl₂) with aflow rate of 1.2 cm/min. During the chromatography process, samples andbuffers were cooled using an ice bath. Finally, the pH of the eluate wasadjusted to 6.9.

Example 13 Conjugation of rFVIII with Dextran

For conjugation of rFVIII with dextran, 2 ml rFVIII (638 mg, 3.4 mg/mlprotein) were transferred to oxidation buffer (50 mM sodium acetate, pH6) using desalting columns (Bio-Rad Econopac 10 DG) according to themanufacturer's instruction. The protein was then oxidated with 0.25 mMNaIO₄ (1 h at 4° C. in the dark). The oxidated protein was firstconcentrated using vivaspin ultrafiltration spin columns (SartoriusStedim Biotech GmbH) with a MWCO of 30 kDa according to themanufacturer's instructions. The sample was next dialyzed againstreaction buffer (50 mM sodium phosphate pH 7) over night at 4° C.

After dialysis, 26.58 mg adipic acid dihydrazide (ADH) (Sigma) was added(500 fold molar excess) and the reaction mixture was incubated 2 h at RTat pH 7 under gentle shaking. ADH was removed using desalting columns(Bio-Rad Econopac 10 DG) according to the instructions of themanufacturer. Ten mg aldehyde-activated dextran (Pierce) was added (17fold molar excess to protein) and the mixture was incubated for 2 h atRT, pH 7.

The conjugate was purified by IEX chromatography on Q-Sepharose HP(GE-Healthcare). The sample was loaded onto a column (6.4 mm×3 cm, V=1ml) that was equilibrated with buffer A (50 mM sodium phosphate pH 6.8)with a flow rate of 0.5 ml/min. Unbound sample was washed out with 5 CVbuffer A. Finally, the conjugate was eluted with a linear salt gradient(0-100% buffer B [50 mM sodium phosphate pH 6.8+1M NaCl] in 10 CV) witha flow rate of 0.5 ml/min.

1. A unit dose of a pharmaceutical composition effective to maintain aFVIII molecule at a level of at least 1% in a patient suffering from ableeding disorder, comprising (i) a conjugate comprising a native FactorVIII modified with a water soluble polymer bound thereto, said watersoluble polymer attached to the modified Factor VIII via an oxidizedcarbohydrate moiety, said modified Factor VIII retaining at least 50% ofnative FVIII activity, and (ii) a pharmaceutically acceptable excipient.2. The unit dose of claim 1, wherein said composition is substantiallyfree from albumin.
 3. The unit dose of claim 1, wherein said watersoluble polymer is attached to said oxidized carbohydrate moiety via alinker.
 4. The unit dose of claim 3, wherein said linker is4-[4-N-malcimidophenyl]butyric acid hydrazide (MBPH).
 5. The compositionof claim 3, wherein the linker is releasable or hydrolysable.
 6. Thecomposition of claim 1, wherein said oxidized carbohydrate moiety islocated in a native Factor VIII B domain.
 7. The unit dose of claim 1,wherein the pharmaceutical composition is housed in a vessel.
 8. Theunit dose of claim 1, wherein the pharmaceutical composition is housedin a syringe.
 9. The unit dose of claim 1, in a form suited forinjection.
 10. The unit dose of claim 9, wherein the composition is in apowder form for reconstitution with a diluent prior to injection. 11.The unit dose of claim 10, reconstituted with a diluent.
 12. The unitdose of claim 1, wherein the composition comprises a plurality ofconjugates, each having at least one water soluble polymer bound to anative Factor VIII, said water soluble polymer attached to the modifiedFactor VIII via one or more oxidized carbohydrate moieties, saidmodified Factor VIII retaining at least 50% of native FVIII activity,and (ii) a pharmaceutically acceptable excipient.
 13. The unit dose ofclaim 1, further comprising an unconjugated Factor VIII moiety.
 14. Theunit dose of claim 1, wherein each water-soluble polymer in thecomposition has a nominal average molecular weight in a range of fromabout greater than 5,000 Daltons to about 150,000 Daltons.
 15. The unitdose of claim 1, wherein each water-soluble polymer in the compositionis selected from the group consisting of polyethylene glycol (PEG),polysialic acid (PSA) and dextran.
 16. The unit dose of claim 15,wherein each conjugate in the composition comprises the samewater-soluble polymer.
 17. The unit dose of claim 16, wherein thewater-soluble polymer has a nominal average molecular weight in therange of from about 2,000 Daltons to about 150,000 Daltons.
 18. The unitdose of claim 16, wherein the water-soluble polymer has a nominalaverage molecular weight in the range of from about 5,000 Daltons toabout 50,000 Daltons.
 19. The unit dose of claim 16, wherein eachwater-soluble polymer is linear.
 20. The unit dose of claim 16, whereineach water-soluble polymer is branched.
 21. The unit dose of claim 16,wherein the Factor VIII is recombinant Factor VIII.
 22. The unit dose ofclaim 16, wherein the Factor VIII is plasmatic Factor VIII.
 23. A kitcomprising a unit dose of a pharmaceutical composition according toclaim 1 comprising (i) a conjugate comprising a native Factor VIIImodified with a water soluble polymer bound thereto, said water solublepolymer attached to the modified Factor VIII via an oxidizedcarbohydrate moiety, said modified Factor VIII retaining at least 50% ofnative FVIII activity, and (ii) a pharmaceutically acceptable excipient;packaged in a container with a label that describes use of thepharmaceutical composition.
 24. A kit according to claim 23, wherein thepharmaceutical composition is packaged in a unit dose form.
 25. A kitcomprising a unit dose of a pharmaceutical composition according toclaim 1, said kit comprising a first container comprising a conjugatecomprising a native Factor VIII modified with a water soluble polymerbound thereto, said water soluble polymer attached to the modifiedFactor VIII via an oxidized carbohydrate moiety, said modified FactorVIII retaining at least 50% of native FVIII activity, and a secondcontainer comprising a physiologically acceptable reconstitutionsolution for said conjugate in the first container, wherein said kit ispackaged with a label that describes use of the pharmaceuticalcomposition.
 26. A kit according to claim 25, wherein the pharmaceuticalcomposition is packaged in a unit dose form.